Chemical Reporters for the Analysis of Lysine Methylation in Epigenetics The transmission of biological information over generations is regulated beyond the level of the DNA sequence. This fundamental principle has been observed in many biological contexts and has been termed "epigenetics", which begins to describe the basic contribution of environment to phenotype under a given genetic background [1]. At the heart of epigenetic mechanisms is regulated gene expression, which manifests itself in the fine control of nucleosome structure and function, the core unit of chromosomes. One of the key factors that modulate transcription is the posttranslational modification (PTM) of chromatin-associated proteins (histones) [2]. In particular, dynamic lysine methylation of histone tails appears to play essential roles in regulating gene expression and epigenetic phenomena [3, 4]. The families of enzymes that regulate lysine methylation on histones, lysine methyltransferases (KMTs) and lysine demethylases (KDMs), have now been identified and are associated with transcriptional regulation, x-chromosomal inactivation and heterochromatin formation [3, 4]. Interestingly, lysine methylation on several non-histone proteins has also been recently described, which has raised many questions regarding the specificity of KMTs and KDMs as well as their discrete roles in epigenetics [2-5]. Unfortunately, the lack of general methods to characterize lysine methylation on proteins and their respective enzymes has hindered a more general understanding of how this PTM regulates signal transduction and epigenetic mechanisms. To fully appreciate the roles of lysine methylation in biological pathways, new methods with higher sensitivity and generality are required. We therefore propose to develop chemical reporters for lysine methylation that will enable rapid detection and identification of methylated proteins in complex mixtures using bioorthogonal labeling methods (Aim 1). Furthermore, we will develop orthogonal enzyme-substrate pairs to identify selective protein substrates of individual lysine methyltransferases (Aim 2). These studies should uncover novel lysine-methylated proteins and identify specific enzymes that regulate their function in fundamental cellular pathways and epigenetic mechanisms of phenotypic inheritance. Ultimately, these tools should provide a more general understanding of protein methylation in normal physiology and disease. PUBLIC HEALTH RELEVANCE: The modulation of phenotypes by epigenetic mechanisms is central to many biological processes and diseases. A detail understanding of the underlying mechanisms that control epigenetics is therefore essential to human health. Reversible lysine methylation on proteins has emerged as an important PTM that regulates the inheritance of phenotypes, however, the analysis of lysine methylation requires more general methods to characterize specific substrates for KMTs and KDMs. To address this problem, this proposal describes the development of chemical reporters to identify lysine-methylated proteins and specific substrates of KMTs. If successful, these chemical approaches would provide the scientific community with a new set of tools to analyze the role of protein methylation in fundamental cellular pathways and in epigenetic processes.